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Long-term intake of egg white hydrolysate attenuates the development of hypertension in spontaneously hypertensive rats
Life Sciences 78 (2006) 2960 – 2966 www.elsevier.com/locate/lifescie
Long-term intake of egg white hydrolysate attenuates the development of hypertension in spontaneously hypertensive rats Marta Miguel a, Rosina Lo´pez-Fandin˜o a, Mercedes Ramos a, Amaya Aleixandre b,* b
a Instituto de Fermentaciones Industriales (CSIC), Madrid, Spain Instituto de Farmacologı´a y Toxicologı´a (CSIC), Departamento de Farmacologı´a, Facultad de Medicina, U. Complutense, Madrid, Spain
Received 11 August 2005; accepted 25 November 2005
Abstract This paper evaluates the effect of the long-term intake of a hydrolysate of egg white with pepsin (HEW), with a potent angiotensin converting enzyme inhibitory activity, on the development of hypertension of spontaneously hypertensive rats (SHR). After being weaned, male 3-week-old SHR were randomly divided into five groups that were given until the 20th week of life the following drinking fluids: (1) tap water, (2) non-treated egg white 1 g/kg/day, (3) captopril 100 mg/kg/day, (4) HEW 0.5 g/kg/day, and (5) HEW 1 g/kg/day. From the 20th to 25th week of life, animals from all groups were given tap water. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured weekly in the rats, from the 6th to 25th week of life, by the tail cuff method. Development of hypertension was attenuated in the groups treated with captopril and HEW ( P < 0.001 vs. the group that drunk tap water). At the 20th week of life, the arterial blood pressure values of the different groups of rats were: tap water (SBP= 219.5 T 5.7, DBP= 167 T 3.7), non-treated egg white (SBP= 206.4 T 1.43, DBP= 166.4 T 4.9), captopril (SBP= 131.7 T 2.74, DBP= 91.5 T 1.62), HEW 0.5 g/kg/day (SBP= 182.9 T 4.64, DBP= 127.5 T 2.1) and HEW 1 g/kg/day (SBP= 177.7 T 4.72, DBP= 120.1 T 2.4). SBP and DBP increased in the treated SHR when the corresponding antihypertensive treatment was removed. In spite of this, SBP remained lower in the SHR that had received captopril and HEW than in the SHR of the control groups ( P < 0.05). The present results suggest that HEW could be used as a functional food with antihypertensive activity. D 2005 Elsevier Inc. All rights reserved. Keywords: Egg white hydrolysate; Blood pressure; Spontaneously hypertensive rats
Introduction Essential hypertension is considered an important disease that must be treated because it is a well-known risk factor for cerebro- and cardiovascular disorders. Angiotensin-converting enzyme (ACE) is critical for blood pressure control and specific inhibitors of this enzyme are used as antihypertensive drugs. ACE-inhibitory peptides can be obtained by enzymatic hydrolysis of food proteins (Maruyama et al., 1985; Kohama et al., 1988; Yamamoto, 1997). It has been demonstrated that certain food peptides can control arterial blood pressure in hypertensive animals after single oral doses (Nakamura et al., 1995; Fujita et al., 2000; Muguerza et al., 2006) and after long* Corresponding author. Dpto. de Farmacologia, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain. Tel.: +34 91 3941475; fax: +34 91 3941463. E-mail address: [email protected] (A. Aleixandre). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.11.025
term administration (Sipola et al., 2001; Yoshii et al., 2001; Yang et al., 2004; Miguel et al., 2005b). In addition, some of the food derived peptides with ACE-inhibitory activity effectively reduced systolic blood pressure (SBP) and diastolic blood pressure (DBP) in hypertensive patients (Hata et al., 1996; Kawasaki et al., 2002; Seppo et al., 2003). The hydrolysate obtained when egg white was treated with pepsin (HEW) exhibited ACE-inhibitory activity in vitro (Miguel et al., 2004). This hydrolysate decreased arterial blood pressure in spontaneously hypertensive rats (SHR) after short treatment (Miguel et al., 2005a). Some peptides identified in HEW and, in particular, the sequences Tyr-Ala-Glu-Glu-ArgTyr-Pro-Ile-Leu (YAEERYPIL), Arg-Ala-Asp-His-Pro-PheLeu (RADHPFL), and Ile-Val-Phe (IVF), also showed considerable ACE-inhibitory activity in vitro, and antihypertensive acute effects in SHR. Nevertheless, HEW and these peptides did not modify the arterial blood pressure of normotensive Wistar –Kyoto rats (Miguel et al., 2004, 2005a).
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The aim of this study was to evaluate the effect of the longterm intake of HEW on the development of hypertension of SHR. In order to facilitate the interpretation of the results, we have also measured the weight gain and consumption of freely accessible solid feed and drinking fluid in the animals throughout the experimental period.
animals from the different groups throughout the experimental period. All the above-mentioned experiments were performed as authorized for scientific research (European Directive 86/609/ CEE and Royal Decree 223/1988 of the Spanish Ministry of Agriculture, Fisheries and Food).
Methods and materials
Statistical analysis
Products
The results are expressed as mean values T S.E.M. for a minimum of 6 rats, and were analyzed by a two-way analysis of variance (ANOVA), using the GraphPad Prism software. We considered, on the one hand, the treatment period (from weaning until the 20th week of life) and, on the other hand, the follow-up period (from the 21st to the 25th week of life). In addition, in order to compare the different treatments and to assess the effect of time within each treatment, some data were also analyzed by a one-way ANOVA, and differences between the groups were assessed by the Bonferroni test. Differences between the means was considered to be significant when P < 0.05.
Crude egg white was obtained in our laboratory from fresh chicken shell eggs, and HEW was prepared as previously described (Miguel et al., 2004). Captopril (Sigma, USA) was also used in this study. Protocol in rats and blood pressure measurement After being weaned at 3 weeks, male SHR (Charles River Laboratories Espan˜a S.A.) were caged in groups of five rats at a temperature of 23 -C with 12 h light / dark cycles. They were in turn randomly divided with ad libitum intake into five groups of animals. Until the rats were 20 weeks old, the drinking fluids in these groups were as follows: tap water (negative control 1), non-treated egg white 1 g/kg/day (negative control 2), captopril 100 mg/kg/day (positive control), HEW 0.5 g/kg/day and HEW 1 g/kg/day. These doses were selected taking into account previous studies that evaluated the effect of single oral administrations of captopril and HEW to SHR (Miguel et al., 2005a). The drinking fluid was always tap water in all groups of animals from the 20th to 25th week of life. During the experimental period, the SHR of the five established groups were fed on a solid standard diet for rats (A04 Panlab, Barcelona, Spain). In view of the difficulty involved in measuring the arterial blood pressure in very young rats, in this study SBP and DBP were measured weekly in the rats from the 6th to 25th week of life, by the tail cuff method (Bun˜ag, 1973). The original technique for measuring arterial blood pressure using the tail cuff method provides only SBP values, but the equipment used in this study, LE 5001 (Letica, Hospitalet, Barcelona, Spain), has a high sensitivity pulse transducer coupled with an accurate microprocessor program, and allows us to distinguish between SBP and DBP. Before the measurements, the rats were kept at 38 -C for 10 –15 min to make the pulsations of the tail artery detectable. Arterial blood pressure measurements were performed at the same time of day (between 9 a.m. and 13 p.m.) in order to avoid the influence of the circadian cycle, and the values of SBP and DBP were obtained by estimating the average reading of three measurements. Moreover, the person who measured the arterial blood pressure in the animals from the different groups did not know the drinking fluid that had been administered to each of these groups. We took weekly measurements of rat weight up to the 25th week of life in the different groups. Intake of drinking fluids and freely accessible feed was also estimated weekly in the
Results Figs. 1 and 2 show, respectively, the evolution of SBP and DBP of SHR taking the different treatments. The two-way ANOVA of the data revealed that the main factors, treatment and time, contributed to statistical differences in SBP and DBP ( P < 0.01) during the treatment and the follow-up period. The one-way ANOVA showed that no differences were observed in the SBP and DBP values between the SHR that drank tap water and the solution of non-treated egg white. SHR of the negative control groups underwent a gradual increase in SBP and DBP from weaning that reached maximal values at about 17 weeks of life ( P < 0.05). From this age, the SBP and DBP values of these rats remained constantly high, and were similar between weeks 17 and 25. From the 6th to the 20th week of life, the lowest values of SBP and DBP were observed in the rats from the positive control group treated with captopril. Significant differences were observed when these values were compared with the corresponding values in the other groups ( P < 0.001). The animals treated with HEW also showed lower values of SBP and DBP than the animals from the negative control groups ( P < 0.001). There was no statistical difference between the rats treated with a different dose of HEW when we compared the corresponding SBP and DBP values (Figs. 1 and 2). An increase in SBP and DBP was observed in the SHR that had been treated with HEW when these rats were given tap water as drinking fluid from the 20th week of life. However, during the follow-up period, the SBP of these groups of animals remained lower than that of the tap water group ( P < 0.05). SBP and DBP also increased to some extent in the SHR that had received captopril when we interrupted the pharmacological treatment, however these variables were always the lowest among all the experimental groups ( P < 0.001) (Figs. 1 and 2).
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M. Miguel et al. / Life Sciences 78 (2006) 2960 – 2966 Tap water Captopril Non-treated egg white
250
HEW (0.5 g/kg/day)
225
HEW (1 g/kg/day)
200
SBP (mm Hg)
175 150 125 100 75 50
- a,b - a,b,c - a,b,c
25
- a,b - a,c - a,c
0 5
6
7
8
9
10
11 12
13 14 15
16
17 18 19
20 21 22 23 24
25
Weeks of life Fig. 1. Systolic blood pressure (SBP) of spontaneously hypertensive rats. The animals drank different fluids from weaning until the 20th week of life (treatment period indicated by a solid bar). All rats drank tap water from the 20th week of life until the 25th week of life (follow-up period indicated by an open bar). Data are mean values T S.E.M. for 6 – 8 rats. The figures indicate significant differences between the groups of rats in the established periods. aP < 0.05 vs. tap water; bP < 0.05 vs. non-treated egg white; cP < 0.05 vs. captopril. P estimated by one-way ANOVA.
The body weight of the SHR increased progressively from weaning ( P < 0.001), with this increase being more accentuated during the first 11th weeks of life (Fig. 3). The body weight was very similar in all animal groups, although the weight gain was lower in the animals treated with captopril during the follow-up period as compared with the other groups ( P < 0.01). Dry and liquid food intake increased progressively in the SHR of the different groups from the beginning of the experimental period ( P < 0.05). No differences were appreci-
ated when the dry food intake of the animals from the different groups was compared over the entire experimental period (Fig. 4). However, there were differences in liquid diet intake in the animals from the different treated groups ( P < 0.001) (Fig. 5). During the treatment period, the highest liquid diet intake was observed in the SHR treated with captopril or with 1 g/kg/day of HEW, and the lowest liquid diet intake was observed in the rats that drank tap water. The rats from the treated groups showed a clear decrease in liquid diet intake when the
Tap water Captopril Non-treated egg white
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HEW (0.5 g/kg/day)
225
HEW (1 g/kg/day)
DBP (mm Hg)
200 175 150 125 100 75
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50 25
- a,b -c -c
0 5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Weeks of life Fig. 2. Diastolic blood pressure (DBP) of spontaneously hypertensive rats. The animals drank different fluids from weaning until the 20th week of life (treatment period indicated by a solid bar). All rats drank tap water from the 20th week of life until the 25th week of life (follow-up period indicated by an open bar). Data are mean values T S.E.M. for 6 – 8 rats. The figures indicate significant differences between the groups of rats in the established periods. aP < 0.05 vs. tap water; bP < 0.05 vs. non-treated egg white; cP < 0.05 vs. captopril. P estimated by one-way ANOVA.
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× Tap water
Body weight (g)
● Captopril ▲ Non-treated egg white 400 ◆ HEW (0.5 g/kg/day) 375 ■ HEW (1 g/kg/day) 350 325 300 275 250 225 200 175 150 125 100 75 50 25 0 3 4 5 6 7 8
● -a,b ◆ -c 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Weeks of life Fig. 3. Body weight of spontaneously hypertensive rats. The animals drank different fluids from weaning until the 20th week of life (treatment period indicated by a solid bar). All rats drank tap water from the 20th week of life until the 25th week of life (follow-up period indicated by an open bar). Data are mean values T S.E.M. for 6 – 8 rats. The figures indicate significant differences between the groups of rats in the established periods. aP < 0.05 vs. tap water; bP < 0.05 vs. non-treated egg white; cP < 0.05 vs. captopril. P estimated by one-way ANOVA.
treatments were removed and, between the 21st and the 25th week of life, the maximal liquid diet intake was observed in the tap water group that had not been treated. Discussion Several researchers, including our group, have described that before arterial blood pressure stabilizes in SHR, there is an initial period in the life of these animals in which this variable
clearly increases (Sa´nchez et al., 1986; Cabassi et al., 1997; Lo´pez-Miranda et al., 1998; Civantos et al., 1999). An initial gradual increase in SBP and DBP was observed in this study in the SHR of control groups, but the intake of captopril, and also the intake of HEW, attenuated the development of hypertension in these animals. It was not surprising to observe the lowest values of SBP and DBP during the experimental period in the captopril group, because this drug is a well known ACEinhibitor with clinical use in hypertensive patients. Ferrone and
× Tap water Captopril Non-treated egg white
26
HEW (0.5 g/kg/day)
24
HEW (1 g/kg/day)
22
Dry food (g/rat/day)
20 18 16 14 12 10 8 6 4 2 0 2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Weeks of life Fig. 4. Solid diet intake of spontaneously hypertensive rats. The animals drank different fluids from weaning until the 20th week of life (treatment period indicated by a solid bar). All rats drank tap water from the 20th week of life until the 25th week of life (follow-up period indicated by an open bar). Data are mean values T S.E.M. for 6 – 8 rats.
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× Tap water Captopril Non-treated egg white
45
HEW (0.5 g/kg/day) HEW (1 g/kg/day)
Liquid intake (ml/rat/day)
40 35 30 25 20 15 10
- a,b - a,b - a,b
-a -a
5 0 3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Weeks of life Fig. 5. Liquid diet intake of spontaneously hypertensive rats. The animals drank different fluids from weaning until the 20th week of life (treatment period indicated by a solid bar). All rats drank tap water from the 20th week of life until the 25th week of life (follow-up period indicated by an open bar). Data are mean values T S.E.M. for 6 – 8 rats. The figures indicate significant differences between the groups of rats in the established periods. aP < 0.05 vs. tap water; bP < 0.05 vs. non-treated egg white. P estimated by one-way ANOVA.
Antonaccio (1979) treated male weanling SHR for 16 weeks with the same dose of captopril used in this study (100 mg/kg/ day) and also observed that this drug was able to completely prevent the development of hypertension. More recently, Berecek et al. (2005) observed that treatment with this dose of captopril from in utero also had an antihypertensive effect in 12 –14 month old male SHR that was even noticeable in these animals even when the therapy was stopped at weaning. The effect of HEW can be related to the ACE inhibitory activity of the peptides released by pepsin (Miguel et al., 2004), but we cannot exclude that other mechanisms may contribute to the antihypertensive activity. The protective effect exerted by ACE-inhibitors on the structure and functions of the cardiovascular system, the kidney, and the brain, seems also to be related to an antioxidant action and a reduced formation of reactive oxygen species (Basso et al., 2005), and it is noteworthy that HEW and some of its peptides have shown antioxidant effects in vitro (Da´valos et al., 2004). The antihypertensive peptides present in HEW could also exert a vasorelaxing activity. In fact, ovokinin, the first example of bioactive peptide derived from egg protein, showed a relaxing activity mediated by the bradykinin B1 receptor and the release of prostaglandin I2 from the endothelium (Fujita et al., 1995). Moreover, the sequence RADHPFL, present in HEW, is very similar to ovokinin and to an antihypertensive peptide derived from ovokinin, named ovokinin (2-7), that could lower arterial blood pressure through central nervous system effects and through the interaction with receptors expressed in the gastrointestinal tract (Yamada et al., 2002). Long-term administration of different hydrolysates obtained from food proteins by other researchers has also shown antihypertensive properties in
SHR (Sipola et al., 2001; Yang et al., 2004). Yoshii et al. (2001) reported, in particular, that long-term administration of a hydrolysate obtained by enzymatic treatment of egg yolk attenuated the development of hypertension in these animals. In this study, the SBP in the SHR that drank the solution of non-treated egg white was slightly, although not significantly, lower than the SBP in the SHR that drank tap water. Hydrolysis by pepsin in the stomach probably does not lead to the same end-products as the enzymatic hydrolysis carried out in our laboratory, but we cannot rule out the possibility that some antihypertensive peptides may also be produced in vivo as a consequence of the gastrointestinal digestion of egg white proteins. It is also important to bear in mind that egg white is rich in proteins, and that diets rich in proteins decrease arterial blood pressure (Obarzanek et al., 1996). Furthermore, egg white is rich in minerals such as magnesium, potassium and calcium that have also shown antihypertensive properties (McCarron, 1982; Kotchen and Mc Carron, 1998; Civantos and Aleixandre, 2004; Miguel et al., 2005b). After withdrawal of the treatments, arterial blood pressure rose gradually in the rats that had drunk the solutions of HEW, although SBP remained consistently lower than in the water control group. Different researchers have similarly described a gradual rise in SBP when the intake of fermented milks with ACE-inhibitory and antihypertensive activity was stopped in SHR (Sipola et al., 2001; Miguel et al., 2005b). We also observed an increase in the arterial blood pressure of the rats that had been treated with captopril when this treatment was removed. The reversion was, naturally, less noticeable, because this drug is a potent ACE-inhibitor. Kost et al. (2000) also reported a slight increase in arterial blood pressure in SHR following the withdrawal of captopril treatment.
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SHR receiving captopril gained weight at a lower rate than the other animals, and this agrees with previous studies showing that blockade of ACE slows body weight gain in this rat strain (Chow et al., 1997). Liquid consumption increased in the SHR group that drank the most concentrated HEW solution. The higher acceptability of this hydrolysate by the animals could be related to its peptide content. Proteins in their natural state, do not contribute to the flavour of food, but products derived from them, such as amino acids and peptides, do have their own flavour, and, according to Di Nicolantonio (2004), SHR have an exaggerated preference for sweet and salty solutions. Liquid consumption also increased markedly in the rats that drank the captopril solution. This is not surprising because it has been clearly demonstrated that ACE-inhibitors induce thirst and increase water intake and urine output in rats (Barney et al., 1980; Fregly and Rowland, 1991). The ACEinhibitory activity could also account for the higher liquid intake observed in the group treated with 1g/kg/day of HEW with respect to the water group. In conclusion, the hydrolysis of egg white with pepsin may be a successful procedure to produce a functional food with antihypertensive activity. We would like to emphasize, that the slight and maintained effect produced by HEW in our study, may be a clear advantage for its use in hypertensive subjects. Blood pressure variability may contribute to organ damage and is, in fact, a risk factor for cardiovascular diseases (Cruickshank et al., 1987; Psaty et al., 1995; Parati and Mancia, 2001; Pringle et al., 2003). However, we are aware that before routine clinical use of HEW, it would be necessary to carry out clinical studies to demonstrate their long-term antihypertensive efficiency in humans. Acknowledgements This study was supported by the project (AGL2004-06903C02-00). We also thank Manuel Bas Caro, Technician in Pharmacology, for his excellent care of the rats and control of the diets in the different groups of animals. References Barney, C.C., Katovich, M.J., Fregly, M.J., 1980. The effect of acute administration of an angiotensin converting enzyme inhibitor, captopril (SQ 14,225), on experimentally induced thirst in rats. The Journal of Pharmacology and Experimental Therapeutics 12, 53 – 57. Basso, N., Paglia, N., Stella, I., de Cavanagh, E.M.V., Ferder, L., Lores Arnaiz, M.R., Inserra, F., 2005. Protective effect of the inhibition of the rennin – angiotensin system on aging. Regulatory Peptides 128, 247 – 252. Berecek, K.H., Reaves, P., Raizada, M., 2005. Effects of early perturbation of the rennin – angiotensin system on cardiovascular remodelling in spontaneously hypertensive rats. Vascular Pharmacology 42, 93 – 98. Bun˜ag, R.D., 1973. Validation in awake rats of a tail-cuff method for measuring systolic pressure. Journal Applied of Physiology 34, 279 – 282. Cabassi, A., Vinci, S., Calzolari, M., Bruschi, G., Cavatorta, A., Borghetti, A., 1997. The relationship between regional sympathetic activity and the onset of arterial hypertension in spontaneously hypertensive rats. Cardiologia 42, 393 – 396. Chow, L., De Gasparo, M., Levens, M., 1997. Blockade of angiotensin converting enzyme but not of angiotensin AT1 receptors improves glucose tolerance. European Journal of Pharmacology 319, 77 – 83.
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Long-term intake of egg white hydrolysate attenuates the development of hypertension in spontaneously hypertensive rats Marta Miguel a, Rosina Lo´pez-Fandin˜o a, Mercedes Ramos a, Amaya Aleixandre b,* b
a Instituto de Fermentaciones Industriales (CSIC), Madrid, Spain Instituto de Farmacologı´a y Toxicologı´a (CSIC), Departamento de Farmacologı´a, Facultad de Medicina, U. Complutense, Madrid, Spain
Received 11 August 2005; accepted 25 November 2005
Abstract This paper evaluates the effect of the long-term intake of a hydrolysate of egg white with pepsin (HEW), with a potent angiotensin converting enzyme inhibitory activity, on the development of hypertension of spontaneously hypertensive rats (SHR). After being weaned, male 3-week-old SHR were randomly divided into five groups that were given until the 20th week of life the following drinking fluids: (1) tap water, (2) non-treated egg white 1 g/kg/day, (3) captopril 100 mg/kg/day, (4) HEW 0.5 g/kg/day, and (5) HEW 1 g/kg/day. From the 20th to 25th week of life, animals from all groups were given tap water. Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured weekly in the rats, from the 6th to 25th week of life, by the tail cuff method. Development of hypertension was attenuated in the groups treated with captopril and HEW ( P < 0.001 vs. the group that drunk tap water). At the 20th week of life, the arterial blood pressure values of the different groups of rats were: tap water (SBP= 219.5 T 5.7, DBP= 167 T 3.7), non-treated egg white (SBP= 206.4 T 1.43, DBP= 166.4 T 4.9), captopril (SBP= 131.7 T 2.74, DBP= 91.5 T 1.62), HEW 0.5 g/kg/day (SBP= 182.9 T 4.64, DBP= 127.5 T 2.1) and HEW 1 g/kg/day (SBP= 177.7 T 4.72, DBP= 120.1 T 2.4). SBP and DBP increased in the treated SHR when the corresponding antihypertensive treatment was removed. In spite of this, SBP remained lower in the SHR that had received captopril and HEW than in the SHR of the control groups ( P < 0.05). The present results suggest that HEW could be used as a functional food with antihypertensive activity. D 2005 Elsevier Inc. All rights reserved. Keywords: Egg white hydrolysate; Blood pressure; Spontaneously hypertensive rats
Introduction Essential hypertension is considered an important disease that must be treated because it is a well-known risk factor for cerebro- and cardiovascular disorders. Angiotensin-converting enzyme (ACE) is critical for blood pressure control and specific inhibitors of this enzyme are used as antihypertensive drugs. ACE-inhibitory peptides can be obtained by enzymatic hydrolysis of food proteins (Maruyama et al., 1985; Kohama et al., 1988; Yamamoto, 1997). It has been demonstrated that certain food peptides can control arterial blood pressure in hypertensive animals after single oral doses (Nakamura et al., 1995; Fujita et al., 2000; Muguerza et al., 2006) and after long* Corresponding author. Dpto. de Farmacologia, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain. Tel.: +34 91 3941475; fax: +34 91 3941463. E-mail address: [email protected] (A. Aleixandre). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.11.025
term administration (Sipola et al., 2001; Yoshii et al., 2001; Yang et al., 2004; Miguel et al., 2005b). In addition, some of the food derived peptides with ACE-inhibitory activity effectively reduced systolic blood pressure (SBP) and diastolic blood pressure (DBP) in hypertensive patients (Hata et al., 1996; Kawasaki et al., 2002; Seppo et al., 2003). The hydrolysate obtained when egg white was treated with pepsin (HEW) exhibited ACE-inhibitory activity in vitro (Miguel et al., 2004). This hydrolysate decreased arterial blood pressure in spontaneously hypertensive rats (SHR) after short treatment (Miguel et al., 2005a). Some peptides identified in HEW and, in particular, the sequences Tyr-Ala-Glu-Glu-ArgTyr-Pro-Ile-Leu (YAEERYPIL), Arg-Ala-Asp-His-Pro-PheLeu (RADHPFL), and Ile-Val-Phe (IVF), also showed considerable ACE-inhibitory activity in vitro, and antihypertensive acute effects in SHR. Nevertheless, HEW and these peptides did not modify the arterial blood pressure of normotensive Wistar –Kyoto rats (Miguel et al., 2004, 2005a).
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The aim of this study was to evaluate the effect of the longterm intake of HEW on the development of hypertension of SHR. In order to facilitate the interpretation of the results, we have also measured the weight gain and consumption of freely accessible solid feed and drinking fluid in the animals throughout the experimental period.
animals from the different groups throughout the experimental period. All the above-mentioned experiments were performed as authorized for scientific research (European Directive 86/609/ CEE and Royal Decree 223/1988 of the Spanish Ministry of Agriculture, Fisheries and Food).
Methods and materials
Statistical analysis
Products
The results are expressed as mean values T S.E.M. for a minimum of 6 rats, and were analyzed by a two-way analysis of variance (ANOVA), using the GraphPad Prism software. We considered, on the one hand, the treatment period (from weaning until the 20th week of life) and, on the other hand, the follow-up period (from the 21st to the 25th week of life). In addition, in order to compare the different treatments and to assess the effect of time within each treatment, some data were also analyzed by a one-way ANOVA, and differences between the groups were assessed by the Bonferroni test. Differences between the means was considered to be significant when P < 0.05.
Crude egg white was obtained in our laboratory from fresh chicken shell eggs, and HEW was prepared as previously described (Miguel et al., 2004). Captopril (Sigma, USA) was also used in this study. Protocol in rats and blood pressure measurement After being weaned at 3 weeks, male SHR (Charles River Laboratories Espan˜a S.A.) were caged in groups of five rats at a temperature of 23 -C with 12 h light / dark cycles. They were in turn randomly divided with ad libitum intake into five groups of animals. Until the rats were 20 weeks old, the drinking fluids in these groups were as follows: tap water (negative control 1), non-treated egg white 1 g/kg/day (negative control 2), captopril 100 mg/kg/day (positive control), HEW 0.5 g/kg/day and HEW 1 g/kg/day. These doses were selected taking into account previous studies that evaluated the effect of single oral administrations of captopril and HEW to SHR (Miguel et al., 2005a). The drinking fluid was always tap water in all groups of animals from the 20th to 25th week of life. During the experimental period, the SHR of the five established groups were fed on a solid standard diet for rats (A04 Panlab, Barcelona, Spain). In view of the difficulty involved in measuring the arterial blood pressure in very young rats, in this study SBP and DBP were measured weekly in the rats from the 6th to 25th week of life, by the tail cuff method (Bun˜ag, 1973). The original technique for measuring arterial blood pressure using the tail cuff method provides only SBP values, but the equipment used in this study, LE 5001 (Letica, Hospitalet, Barcelona, Spain), has a high sensitivity pulse transducer coupled with an accurate microprocessor program, and allows us to distinguish between SBP and DBP. Before the measurements, the rats were kept at 38 -C for 10 –15 min to make the pulsations of the tail artery detectable. Arterial blood pressure measurements were performed at the same time of day (between 9 a.m. and 13 p.m.) in order to avoid the influence of the circadian cycle, and the values of SBP and DBP were obtained by estimating the average reading of three measurements. Moreover, the person who measured the arterial blood pressure in the animals from the different groups did not know the drinking fluid that had been administered to each of these groups. We took weekly measurements of rat weight up to the 25th week of life in the different groups. Intake of drinking fluids and freely accessible feed was also estimated weekly in the
Results Figs. 1 and 2 show, respectively, the evolution of SBP and DBP of SHR taking the different treatments. The two-way ANOVA of the data revealed that the main factors, treatment and time, contributed to statistical differences in SBP and DBP ( P < 0.01) during the treatment and the follow-up period. The one-way ANOVA showed that no differences were observed in the SBP and DBP values between the SHR that drank tap water and the solution of non-treated egg white. SHR of the negative control groups underwent a gradual increase in SBP and DBP from weaning that reached maximal values at about 17 weeks of life ( P < 0.05). From this age, the SBP and DBP values of these rats remained constantly high, and were similar between weeks 17 and 25. From the 6th to the 20th week of life, the lowest values of SBP and DBP were observed in the rats from the positive control group treated with captopril. Significant differences were observed when these values were compared with the corresponding values in the other groups ( P < 0.001). The animals treated with HEW also showed lower values of SBP and DBP than the animals from the negative control groups ( P < 0.001). There was no statistical difference between the rats treated with a different dose of HEW when we compared the corresponding SBP and DBP values (Figs. 1 and 2). An increase in SBP and DBP was observed in the SHR that had been treated with HEW when these rats were given tap water as drinking fluid from the 20th week of life. However, during the follow-up period, the SBP of these groups of animals remained lower than that of the tap water group ( P < 0.05). SBP and DBP also increased to some extent in the SHR that had received captopril when we interrupted the pharmacological treatment, however these variables were always the lowest among all the experimental groups ( P < 0.001) (Figs. 1 and 2).
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M. Miguel et al. / Life Sciences 78 (2006) 2960 – 2966 Tap water Captopril Non-treated egg white
250
HEW (0.5 g/kg/day)
225
HEW (1 g/kg/day)
200
SBP (mm Hg)
175 150 125 100 75 50
- a,b - a,b,c - a,b,c
25
- a,b - a,c - a,c
0 5
6
7
8
9
10
11 12
13 14 15
16
17 18 19
20 21 22 23 24
25
Weeks of life Fig. 1. Systolic blood pressure (SBP) of spontaneously hypertensive rats. The animals drank different fluids from weaning until the 20th week of life (treatment period indicated by a solid bar). All rats drank tap water from the 20th week of life until the 25th week of life (follow-up period indicated by an open bar). Data are mean values T S.E.M. for 6 – 8 rats. The figures indicate significant differences between the groups of rats in the established periods. aP < 0.05 vs. tap water; bP < 0.05 vs. non-treated egg white; cP < 0.05 vs. captopril. P estimated by one-way ANOVA.
The body weight of the SHR increased progressively from weaning ( P < 0.001), with this increase being more accentuated during the first 11th weeks of life (Fig. 3). The body weight was very similar in all animal groups, although the weight gain was lower in the animals treated with captopril during the follow-up period as compared with the other groups ( P < 0.01). Dry and liquid food intake increased progressively in the SHR of the different groups from the beginning of the experimental period ( P < 0.05). No differences were appreci-
ated when the dry food intake of the animals from the different groups was compared over the entire experimental period (Fig. 4). However, there were differences in liquid diet intake in the animals from the different treated groups ( P < 0.001) (Fig. 5). During the treatment period, the highest liquid diet intake was observed in the SHR treated with captopril or with 1 g/kg/day of HEW, and the lowest liquid diet intake was observed in the rats that drank tap water. The rats from the treated groups showed a clear decrease in liquid diet intake when the
Tap water Captopril Non-treated egg white
250
HEW (0.5 g/kg/day)
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HEW (1 g/kg/day)
DBP (mm Hg)
200 175 150 125 100 75
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50 25
- a,b -c -c
0 5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Weeks of life Fig. 2. Diastolic blood pressure (DBP) of spontaneously hypertensive rats. The animals drank different fluids from weaning until the 20th week of life (treatment period indicated by a solid bar). All rats drank tap water from the 20th week of life until the 25th week of life (follow-up period indicated by an open bar). Data are mean values T S.E.M. for 6 – 8 rats. The figures indicate significant differences between the groups of rats in the established periods. aP < 0.05 vs. tap water; bP < 0.05 vs. non-treated egg white; cP < 0.05 vs. captopril. P estimated by one-way ANOVA.
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× Tap water
Body weight (g)
● Captopril ▲ Non-treated egg white 400 ◆ HEW (0.5 g/kg/day) 375 ■ HEW (1 g/kg/day) 350 325 300 275 250 225 200 175 150 125 100 75 50 25 0 3 4 5 6 7 8
● -a,b ◆ -c 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Weeks of life Fig. 3. Body weight of spontaneously hypertensive rats. The animals drank different fluids from weaning until the 20th week of life (treatment period indicated by a solid bar). All rats drank tap water from the 20th week of life until the 25th week of life (follow-up period indicated by an open bar). Data are mean values T S.E.M. for 6 – 8 rats. The figures indicate significant differences between the groups of rats in the established periods. aP < 0.05 vs. tap water; bP < 0.05 vs. non-treated egg white; cP < 0.05 vs. captopril. P estimated by one-way ANOVA.
treatments were removed and, between the 21st and the 25th week of life, the maximal liquid diet intake was observed in the tap water group that had not been treated. Discussion Several researchers, including our group, have described that before arterial blood pressure stabilizes in SHR, there is an initial period in the life of these animals in which this variable
clearly increases (Sa´nchez et al., 1986; Cabassi et al., 1997; Lo´pez-Miranda et al., 1998; Civantos et al., 1999). An initial gradual increase in SBP and DBP was observed in this study in the SHR of control groups, but the intake of captopril, and also the intake of HEW, attenuated the development of hypertension in these animals. It was not surprising to observe the lowest values of SBP and DBP during the experimental period in the captopril group, because this drug is a well known ACEinhibitor with clinical use in hypertensive patients. Ferrone and
× Tap water Captopril Non-treated egg white
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HEW (0.5 g/kg/day)
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Dry food (g/rat/day)
20 18 16 14 12 10 8 6 4 2 0 2
3
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7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Weeks of life Fig. 4. Solid diet intake of spontaneously hypertensive rats. The animals drank different fluids from weaning until the 20th week of life (treatment period indicated by a solid bar). All rats drank tap water from the 20th week of life until the 25th week of life (follow-up period indicated by an open bar). Data are mean values T S.E.M. for 6 – 8 rats.
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× Tap water Captopril Non-treated egg white
45
HEW (0.5 g/kg/day) HEW (1 g/kg/day)
Liquid intake (ml/rat/day)
40 35 30 25 20 15 10
- a,b - a,b - a,b
-a -a
5 0 3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Weeks of life Fig. 5. Liquid diet intake of spontaneously hypertensive rats. The animals drank different fluids from weaning until the 20th week of life (treatment period indicated by a solid bar). All rats drank tap water from the 20th week of life until the 25th week of life (follow-up period indicated by an open bar). Data are mean values T S.E.M. for 6 – 8 rats. The figures indicate significant differences between the groups of rats in the established periods. aP < 0.05 vs. tap water; bP < 0.05 vs. non-treated egg white. P estimated by one-way ANOVA.
Antonaccio (1979) treated male weanling SHR for 16 weeks with the same dose of captopril used in this study (100 mg/kg/ day) and also observed that this drug was able to completely prevent the development of hypertension. More recently, Berecek et al. (2005) observed that treatment with this dose of captopril from in utero also had an antihypertensive effect in 12 –14 month old male SHR that was even noticeable in these animals even when the therapy was stopped at weaning. The effect of HEW can be related to the ACE inhibitory activity of the peptides released by pepsin (Miguel et al., 2004), but we cannot exclude that other mechanisms may contribute to the antihypertensive activity. The protective effect exerted by ACE-inhibitors on the structure and functions of the cardiovascular system, the kidney, and the brain, seems also to be related to an antioxidant action and a reduced formation of reactive oxygen species (Basso et al., 2005), and it is noteworthy that HEW and some of its peptides have shown antioxidant effects in vitro (Da´valos et al., 2004). The antihypertensive peptides present in HEW could also exert a vasorelaxing activity. In fact, ovokinin, the first example of bioactive peptide derived from egg protein, showed a relaxing activity mediated by the bradykinin B1 receptor and the release of prostaglandin I2 from the endothelium (Fujita et al., 1995). Moreover, the sequence RADHPFL, present in HEW, is very similar to ovokinin and to an antihypertensive peptide derived from ovokinin, named ovokinin (2-7), that could lower arterial blood pressure through central nervous system effects and through the interaction with receptors expressed in the gastrointestinal tract (Yamada et al., 2002). Long-term administration of different hydrolysates obtained from food proteins by other researchers has also shown antihypertensive properties in
SHR (Sipola et al., 2001; Yang et al., 2004). Yoshii et al. (2001) reported, in particular, that long-term administration of a hydrolysate obtained by enzymatic treatment of egg yolk attenuated the development of hypertension in these animals. In this study, the SBP in the SHR that drank the solution of non-treated egg white was slightly, although not significantly, lower than the SBP in the SHR that drank tap water. Hydrolysis by pepsin in the stomach probably does not lead to the same end-products as the enzymatic hydrolysis carried out in our laboratory, but we cannot rule out the possibility that some antihypertensive peptides may also be produced in vivo as a consequence of the gastrointestinal digestion of egg white proteins. It is also important to bear in mind that egg white is rich in proteins, and that diets rich in proteins decrease arterial blood pressure (Obarzanek et al., 1996). Furthermore, egg white is rich in minerals such as magnesium, potassium and calcium that have also shown antihypertensive properties (McCarron, 1982; Kotchen and Mc Carron, 1998; Civantos and Aleixandre, 2004; Miguel et al., 2005b). After withdrawal of the treatments, arterial blood pressure rose gradually in the rats that had drunk the solutions of HEW, although SBP remained consistently lower than in the water control group. Different researchers have similarly described a gradual rise in SBP when the intake of fermented milks with ACE-inhibitory and antihypertensive activity was stopped in SHR (Sipola et al., 2001; Miguel et al., 2005b). We also observed an increase in the arterial blood pressure of the rats that had been treated with captopril when this treatment was removed. The reversion was, naturally, less noticeable, because this drug is a potent ACE-inhibitor. Kost et al. (2000) also reported a slight increase in arterial blood pressure in SHR following the withdrawal of captopril treatment.
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SHR receiving captopril gained weight at a lower rate than the other animals, and this agrees with previous studies showing that blockade of ACE slows body weight gain in this rat strain (Chow et al., 1997). Liquid consumption increased in the SHR group that drank the most concentrated HEW solution. The higher acceptability of this hydrolysate by the animals could be related to its peptide content. Proteins in their natural state, do not contribute to the flavour of food, but products derived from them, such as amino acids and peptides, do have their own flavour, and, according to Di Nicolantonio (2004), SHR have an exaggerated preference for sweet and salty solutions. Liquid consumption also increased markedly in the rats that drank the captopril solution. This is not surprising because it has been clearly demonstrated that ACE-inhibitors induce thirst and increase water intake and urine output in rats (Barney et al., 1980; Fregly and Rowland, 1991). The ACEinhibitory activity could also account for the higher liquid intake observed in the group treated with 1g/kg/day of HEW with respect to the water group. In conclusion, the hydrolysis of egg white with pepsin may be a successful procedure to produce a functional food with antihypertensive activity. We would like to emphasize, that the slight and maintained effect produced by HEW in our study, may be a clear advantage for its use in hypertensive subjects. Blood pressure variability may contribute to organ damage and is, in fact, a risk factor for cardiovascular diseases (Cruickshank et al., 1987; Psaty et al., 1995; Parati and Mancia, 2001; Pringle et al., 2003). However, we are aware that before routine clinical use of HEW, it would be necessary to carry out clinical studies to demonstrate their long-term antihypertensive efficiency in humans. Acknowledgements This study was supported by the project (AGL2004-06903C02-00). We also thank Manuel Bas Caro, Technician in Pharmacology, for his excellent care of the rats and control of the diets in the different groups of animals. References Barney, C.C., Katovich, M.J., Fregly, M.J., 1980. The effect of acute administration of an angiotensin converting enzyme inhibitor, captopril (SQ 14,225), on experimentally induced thirst in rats. The Journal of Pharmacology and Experimental Therapeutics 12, 53 – 57. Basso, N., Paglia, N., Stella, I., de Cavanagh, E.M.V., Ferder, L., Lores Arnaiz, M.R., Inserra, F., 2005. Protective effect of the inhibition of the rennin – angiotensin system on aging. Regulatory Peptides 128, 247 – 252. Berecek, K.H., Reaves, P., Raizada, M., 2005. Effects of early perturbation of the rennin – angiotensin system on cardiovascular remodelling in spontaneously hypertensive rats. Vascular Pharmacology 42, 93 – 98. Bun˜ag, R.D., 1973. Validation in awake rats of a tail-cuff method for measuring systolic pressure. Journal Applied of Physiology 34, 279 – 282. Cabassi, A., Vinci, S., Calzolari, M., Bruschi, G., Cavatorta, A., Borghetti, A., 1997. The relationship between regional sympathetic activity and the onset of arterial hypertension in spontaneously hypertensive rats. Cardiologia 42, 393 – 396. Chow, L., De Gasparo, M., Levens, M., 1997. Blockade of angiotensin converting enzyme but not of angiotensin AT1 receptors improves glucose tolerance. European Journal of Pharmacology 319, 77 – 83.
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